Project Summary The overarching hypothesis of the Center for Alcohol Research in Epigenetics (CARE) is that epigenetic mechanisms drive alterations in the brain transcriptome after chronic alcohol exposure and withdrawal, leading to neuroadaptations that promote dependence-induced behaviors such as anxiety, depression, and escalated ethanol intake. Research project 3 of CARE has identified aberrant expression of innate immune genes in the hippocampus during withdrawal from chronic alcohol drinking. An increased neuroinflammatory response in the hippocampus during withdrawal may cause depression-related behaviors that promote escalated alcohol intake. Preliminary data suggests that the induction of neuroimmune genes during withdrawal may be driven by increased expression of the histone demethylase KDM6B, which removes transcriptionally-repressive methyl groups from histone H3 lysine 27 (H3K27me3). The upregulation of KDM6B during withdrawal from chronic alcohol is predicted to decrease H3K27me3 at specific gene promoters and increase chromatin accessibility, leading to increased gene expression. The first set of experiments will use unbiased methods to examine changes in chromatin accessibility and H3K27me3 levels at genomic loci in the hippocampus during withdrawal. This will be accomplished using the assay for transposase accessible chromatin and chromatin immunoprecipitation with H3K27me3 antibody followed by DNA sequencing (ATAC-Seq and ChIP-Seq, respectively) in the hippocampus of rats that have been treated with chronic ethanol and withdrawal in a dependence model (Lieber DeCarli ethanol liquid diet). These experiments will be performed in collaboration with the Epigenetics and Behavioral Cores of CARE. The second set of experiments will examine the role of KDM6B and H3K27me3 in the expression of specific neuroimmune gene targets and determine, using viral-mediated RNA interference or a pharmacological inhibitor of KDM6B, the functional role of KDM6B in depression-like behavior during withdrawal. In the third set of experiments, the mechanism of Kdm6b gene induction during alcohol withdrawal will be investigated. Kdm6b expression is known to be regulated by STAT3, which is also increased and activated in the hippocampus during withdrawal from chronic alcohol drinking. Previous studies have shown that STAT3 can interact with the histone deacetylase HDAC6 to regulate transcription. We will examine the regulation of Kdm6b gene expression by STAT3 and HDAC6, and determine if pharmacological inhibition or RNAi-mediated down-regulation of STAT3 or HDAC6 during withdrawal affects depression-like behavior and escalated ethanol intake (in collaboration with the Behavioral Core) during withdrawal. Successful completion of these experiments will further two goals of CARE, which are to delineate epigenetic mechanisms operative in alcohol use disorder and identify new epigenetic targets for development of pharmacotherapy to treat alcohol use disorder.